Optimization, characterization and heterologous expression of clucoamylase and cx-Amylase form Aspergillus flavus NSH9 in Pichia pastoris

Glucoamylase (GA) is an exohydrolases, which release β-glucose units from the non-reducing ends of starch have industrial importance. The purposes of the study were to optimize glucoamylase production by Aspergillus flavus NSH9, to isolate and characterize amylase (glucoamylase and -amylase) gene a...

Full description

Saved in:

Bibliographic Details
Main Author:	Kazi Muhammad, Rezaul Karim
Format:	Thesis
Language:	English
Published:	2016
Subjects:	TP Chemical technology
Online Access:	http://ir.unimas.my/id/eprint/30690/1/Kazi%20ft.pdf
Tags:	Add Tag No Tags, Be the first to tag this record!

id	my-unimas-ir.30690
record_format	uketd_dc
spelling	my-unimas-ir.306902023-10-19T08:58:29Z Optimization, characterization and heterologous expression of clucoamylase and cx-Amylase form Aspergillus flavus NSH9 in Pichia pastoris 2016 Kazi Muhammad, Rezaul Karim TP Chemical technology Glucoamylase (GA) is an exohydrolases, which release β-glucose units from the non-reducing ends of starch have industrial importance. The purposes of the study were to optimize glucoamylase production by Aspergillus flavus NSH9, to isolate and characterize amylase (glucoamylase and -amylase) gene and to clone as well as to characterize the recombinant glucoamylase (rGA) heterologously expressed in Pichia pastoris. The highest activity of crude glucoamylase was observed in the fermentation media after 5 days of incubation at 25°C and at pH 5.0. Crude GA exhibited optimum catalytic activity at pH 5.0 and temperature of 70°C. The molecular weight of the purified -amylase was estimated to be 54 kDa and exhibited optimum catalytic activity at pH 5.0 and temperature of 50°C. Three complete open reading frames of amylase gene from A. flavus were identified; those encoded 493, 612 and 499 amino acids for GA1, GA2 and -amylase; respectively. The first 17, 19 and 21 amino acids were presumed to be the signal peptides for GA1, GA2, and -amylase; respectively. Both of the glucoamylases were assigned to glycosyl hydrolase family 15, and -amylase to glycosyl hydrolase family 13. All three cDNA were cloned into Pichia pastoris. The highest expression of recombinant glucoamylase (rGA1) was observed after 8 days of incubation period with 1% methanol. The molecular weight of the purified rGA1 was about 78 kDa and exhibited optimum catalytic activity at pH 5.0 and temperature of 70°C, and also thermostable. Low concentration of metal had a positive effect on rGA1 activity. GA2 was also successfully expressed and had significant raw starch degrading capacity. This two rGA might be used in the saccharification steps in the starch processing industry, due to its thermostability and raw starch degrading properties Universiti Malaysia Sarawak(UNIMAS) 2016 Thesis http://ir.unimas.my/id/eprint/30690/ http://ir.unimas.my/id/eprint/30690/1/Kazi%20ft.pdf text en validuser phd doctoral Universiti Malaysia Sarawak(UNIMAS) Faculty of Resource Science and Technology
institution	Universiti Malaysia Sarawak
collection	UNIMAS Institutional Repository
language	English
topic	TP Chemical technology
spellingShingle	TP Chemical technology Kazi Muhammad, Rezaul Karim Optimization, characterization and heterologous expression of clucoamylase and cx-Amylase form Aspergillus flavus NSH9 in Pichia pastoris
description	Glucoamylase (GA) is an exohydrolases, which release β-glucose units from the non-reducing ends of starch have industrial importance. The purposes of the study were to optimize glucoamylase production by Aspergillus flavus NSH9, to isolate and characterize amylase (glucoamylase and -amylase) gene and to clone as well as to characterize the recombinant glucoamylase (rGA) heterologously expressed in Pichia pastoris. The highest activity of crude glucoamylase was observed in the fermentation media after 5 days of incubation at 25°C and at pH 5.0. Crude GA exhibited optimum catalytic activity at pH 5.0 and temperature of 70°C. The molecular weight of the purified -amylase was estimated to be 54 kDa and exhibited optimum catalytic activity at pH 5.0 and temperature of 50°C. Three complete open reading frames of amylase gene from A. flavus were identified; those encoded 493, 612 and 499 amino acids for GA1, GA2 and -amylase; respectively. The first 17, 19 and 21 amino acids were presumed to be the signal peptides for GA1, GA2, and -amylase; respectively. Both of the glucoamylases were assigned to glycosyl hydrolase family 15, and -amylase to glycosyl hydrolase family 13. All three cDNA were cloned into Pichia pastoris. The highest expression of recombinant glucoamylase (rGA1) was observed after 8 days of incubation period with 1% methanol. The molecular weight of the purified rGA1 was about 78 kDa and exhibited optimum catalytic activity at pH 5.0 and temperature of 70°C, and also thermostable. Low concentration of metal had a positive effect on rGA1 activity. GA2 was also successfully expressed and had significant raw starch degrading capacity. This two rGA might be used in the saccharification steps in the starch processing industry, due to its thermostability and raw starch degrading properties
format	Thesis
qualification_name	Doctor of Philosophy (PhD.)
qualification_level	Doctorate
author	Kazi Muhammad, Rezaul Karim
author_facet	Kazi Muhammad, Rezaul Karim
author_sort	Kazi Muhammad, Rezaul Karim
title	Optimization, characterization and heterologous expression of clucoamylase and cx-Amylase form Aspergillus flavus NSH9 in Pichia pastoris
title_short	Optimization, characterization and heterologous expression of clucoamylase and cx-Amylase form Aspergillus flavus NSH9 in Pichia pastoris
title_full	Optimization, characterization and heterologous expression of clucoamylase and cx-Amylase form Aspergillus flavus NSH9 in Pichia pastoris
title_fullStr	Optimization, characterization and heterologous expression of clucoamylase and cx-Amylase form Aspergillus flavus NSH9 in Pichia pastoris
title_full_unstemmed	Optimization, characterization and heterologous expression of clucoamylase and cx-Amylase form Aspergillus flavus NSH9 in Pichia pastoris
title_sort	optimization, characterization and heterologous expression of clucoamylase and cx-amylase form aspergillus flavus nsh9 in pichia pastoris
granting_institution	Universiti Malaysia Sarawak(UNIMAS)
granting_department	Faculty of Resource Science and Technology
publishDate	2016
url	http://ir.unimas.my/id/eprint/30690/1/Kazi%20ft.pdf
_version_	1783728391728398336

Optimization, characterization and heterologous expression of clucoamylase and cx-Amylase form Aspergillus flavus NSH9 in Pichia pastoris

Similar Items